Growth of Escherichia coli, a mesophilic bacterium, is limited at elevated temperatures (13, 14). Quite unexpectedly, earlier investigations showed that E. coli did not grow at temperatures above 44° C. because of the instability of a single protein, homoserine o-succinyltransferase (MetA) (13, 14, 15, 16). MetA (EC 2.1.3.46), the first enzyme in methionine biosynthesis (FIG. 1), catalyzes the transfer of succinate from succinyl-CoA to L-homoserine (3, 22). Recent findings report that MetAE.coli tends to unfold even at 25° C. in vitro; unfolding becomes maximal at 44° C. and is followed by massive aggregation (5). In vivo, the soluble fraction of cytoplasmic proteins lacks MetAE.coli at temperatures higher than 44° C. (5). MetA from Salmonella enterica is as sensitive to elevated temperature as to weak organic acids including benzoate, propionate and acetate (11). Moreover, hydrogen peroxide increases its sensitivity to both heat and acid and may oxidatively damage the destabilized MetA (11). Price-Carter and coworkers (11) suggested that an excess of MetAS.enterica synthesized at elevated temperatures and/or in the presence of weak organic acids leads to the accumulation of insoluble aggregates that are toxic to the cells and inhibit bacterial growth.
In view of all the foregoing data and the fact that MetA occupies the control point in methionine biosynthesis, it has been proposed that MetA plays a central role in the control of bacterial growth (2). MetA's high sensitivity to many stress factors suggests that it may serve as a sort of metabolic fuse, detecting unfavorable growth conditions (11). In this connection, it is notable that methionine relieves the inhibitory effect of high-temperature and acetic acid on E. coli growth (7, 12, 13, 14).
However, an attempt to enhance thermostability of E. coli by modifying an enzyme (particularly, MetA) responsible for the synthesis of methionine has not been reported yet. The reason is as follows: (a) it is problematic to enhance thermostability of the enzyme per se by modifying the enzyme of mesophilic bacteria; and (b) it is quite difficult to expect whether its modification enhances the growth rate of bacteria at a high-temperature.
For example, US Pat. Appln. Pub. No. 20050233308 discloses a method for enhancing thermostability of an enzyme by substituting Arg, Thr and Ala residues for Lys, Ser and Ser residues of the protein, respectively. However, this specification predicts only amino acid residues implicated in thermostability of enzymes through orthologs study of mesophilic bacteria (Corynebacterium glutamicum) and thermophilic bacteria (Corynebacterium efficiens) with no practical experiments. In other words, this patent document provide no practical data on enhancing thermostability of enzymes per se via protein modification or evolution, and did not disclose that its modification or evolution permits to enhance the growth rate of bacteria at higher temperatures. Furthermore, this patent never discloses MetA which is a target enzyme of the present invention.
US Pat. Appln. Pub. No. 20020137094 discloses a method for predicting amino acid residues pivotal in protein thermostability via a multiple alignment of protein sequences. This patent document also provides no practical data on enhancing thermostability of enzymes per se via protein modification or evolution, and never discloses that its modification or evolution contributes to enhancing the growth rate of bacteria at higher temperatures.
In these connections, it would be a quite interesting to widen the optimum growth temperatures of E. coli by increasing the stability of the MetA enzyme, which is one of aims of the present invention.
The above information disclosed in this Background Art section is only for enhancement of understanding of the background of the invention and therefore it may contain information that does not form the prior art that is already known in this country to a person of ordinary skill in the art.
Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.